Basic brainstem taste responsivity: effects of perinatal ... · Rubio-Navarro et al. Revista eNeurobiología 2(1):090511, 2011 4 moves its tongue laterally without showing an aversive

Review Article

Basic brainstem taste responsivity: effects of perinatal influences

Respuestas gustativas del tallo cerebral: efectos de influencias perinatales

Lorena Rubio-Navarro*, Carmen Torrero, Manuel Salas.

Department of Developmental Neurobiology and Neurophysiology, Institute of Neurobiology, Universidad

Nacional Autónoma de México, Juriquilla, Querétaro, México

Abstract

In altricial newborns the gustatory system is fundamental for survival and for establishing mother-litter

bonds in the nest environment. The chemosensory experience is initiated in the uterus by the actions of

chemical stimulants in the amniotic fluid. After birth, maternal care prevails, and the gustatory system is

surprisingly enriched by breast milk suction. In Norway rats at 12 days of age pups make a transition from

milk to solid food. At this time, when the gustatory experience is broadly developed, both the receptors and

the central nervous system (CNS) sensory relay systems undergo a remarkable reorganization to permit the

integration of the sensory and hedonic characteristics of the gustatory cues. The current review analyzes the

morphofunctional organization of the taste buds and the afferent projections, the neuronal organization of

the first CNS relay, as well as how perinatal food deprivation interferes with the plastic properties of the

rostral portion of the brainstem solitary tract nucleus in the rat.

Keywords: Gustatory system, Development, Solitary tract, Rats.

Resumen

En los recién nacidos altriciales, el sistema gustativo juega un papel fundamental para la supervivencia, y

establecimiento de nexos con la madre en el contexto del ambiente del nido. La experiencia quimiosensorial

se inicia en el hábitat uterino por la acción de la estimulación química vía del fluido amniótico. Al nacimiento,

los cuidados maternos prevalecen y el sistema gustativo es ahora fortalecido en su función por la succión de

leche materna. En ratas Norway después de los 12 días de edad, las crías de entran a un periodo de

transición entre la leche y la ingesta de alimento sólido. En este momento, la experiencia gustativa se

desarrolla ampliamente, tanto los receptores como los relevos sensoriales del sistema nervioso central

(SNC) están bajo una notoria reorganización que permite la integración de las características sensoriales y

hedónicas de las señales gustativas. En la presente revisión se analizan la organización morfológica y funcional

de los botones gustativos, las proyecciones aferentes, los cambios neuronales en el primer relevo dentro del

SNC y cómo la restricción perinatal de alimento interfiere con las propiedades plásticas de la porción

rostral del núcleo del tracto solitario de ratas Wistar en desarrollo.

Palabras clave: Sistema gustativo, Desarrollo, Tracto solitario, Ratas.

Corresponding Author: M.Sc. Lorena Rubio-Navarro, Department of Developmental Neurobiology and

Neurophysiology, Institute of Neurobiology, Universidad Nacional Autónoma de México, Juriquilla, Querétaro, Qro.,

76230. México. Tel: 52 5556234059. Fax: 52 5556234038. E-mail: [email protected]. Este es un artículo de libre acceso distribuido bajo los términos de la licencia de Creative Commons, (http://creativecommons.org/licenses/by-nc/3.0), que permite el uso no comercial, distribución y reproducción en algún medio,

siempre que la obra original sea debidamente citada.

mailto:[email protected]

http://creativecommons.org/licenses/by-nc/3.0

http://creativecommons.org/licenses/by

Rubio-Navarro et al. Revista eNeurobiología 2(1):090511, 2011

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Contents

I. Introduction

1. Ontogeny of facial responses to taste

2. Taste bud development

3. Development of central nervous system (CNS) afferents and early properties of the

gustatory system

4. The first relay of the gustatory system in the CNS

4.1 Anatomy of the Solitary Tract Nucleus (STN)

4.2 STN afferents and projections

4.3 Types of neurons in the rostral solitary tract nucleus (STNr)

4.4 Neurogenesis and neurochemical development of the STN

4.5 Synaptogenesis in the STNr

4.6 Neurotransmitters involved in STN function

4.7 Gustotopic organization in the STN

5. Early environmental influences on the gustatory system

6. Alterations produced in the STNr by perinatal sodium restriction

7. Alterations in the STNr by perinatal undernutrition

II. Conclusions

III. Acknowledgements

IV. References


3

I. Introduction

Altricial newborns must face numerous and

continuous environmental demands with

remarkably immature sensorial, motor, and

autonomic systems in order to survive with the

help of maternal care. During the neonatal

period the newborn undergoes a series of

complex morphofunctional brain changes in

order to generate neurons, interconnect them,

complete circuits, progressively increase

neuronal interactions, and adapt the

intracellular neuronal machinery to the diverse

transitory or long-term plastic functional

changes occurring throughout the life span. In

mammals, brain development occurs in two

different, predictable environments: the uterine

habitat, in which a direct chemical link between

mother and pup is established by means of the

fetus-placenta circulation; after birth, pups

encounter the nest environment, where

maternal care prevails, and the mother

constitutes the main source of food and

sensory signals.

From several studies it is known that life in

the uterus is constantly modified within a very

narrow range of conditions and that the fetus

is primarily exposed to somatosensory,

vestibular, and chemosensory stimulation in

preparation for a less stable nest environment.

Thus, the altricial newborn must adapt to a

highly variable external habitat with well-

known sensorial, motor, chemosensory, and

homeostatic deficiencies that are only

overcome with intense maternal care.1-4

Among the signals that the fetus receives and

responds to in the uterus are the chemo-

sensorial cues; the gustatory and olfactory

systems in particular begin to develop early in

gestation, are well advanced by the time of

delivery, and undergo a neonatal period of

rapid development.5-8 The developing olfactory

system in the uterus prepares the fetus for

respiration and initiates the transduction of

signals from maternal odor stimuli included in

the amniotic fluid that reach the fetal olfactory

mucous area.9

Initial studies of the gustatory system were

made in sheep, where the deglutition rate of

amniotic fluid was found to change, depending

on its chemical composition. For instance,

when sucrose was injected into the amniotic

fluid, the rate of fetal deglutition was greater

than when a neutral gustatory stimulant was

injected as observed by the reduction of mouth

movements and licking lips. Therefore, it was

proposed that chemical stimulation of the

fetus, via the gustatory cues in the amniotic

fluid, may stimulate deglutition as a mechanism

to provide nutrients and to promote gastro-

intestinal tract development.10

At birth the gustatory experience is

surprisingly rich; thus, with breast milk suction,

the young satisfies two primary needs,

nourishment and fluid balance, during the first

17 days of age. Behavioral studies have

demonstrated that the newborn can distinguish

at least three basic flavors, and that the

gustatory response is modified until the subject

acquires the adult behavioral pattern at the end

of the lactating period, when the young has

free access to solid food.11 From the pioneering

study of Galef and Henderson12 it is known

that at the end of the second postnatal week,

the young rat already has the gustatory ability

to discriminate among chemical cues, which

allows it to obtain essential sensorial

experience through the different components

of mother’s milk.

The present review focuses on studies using

a variety of research tools in order to provide

information about the complex integration of

the gustatory system; we also include data on

the development of gustatory behavior the

basic elements of the gustatory system: taste

buds, afferent fibers carrying the information to

the first CNS relay, the solitary tract nucleus

(STN) and its general anatomic characteristics

at early stages of development.

1. Ontogeny of facial responses to taste

An important tool to assess gustatory

discrimination in the newborn rat has been the

characteristics of facial responses elicited by

exposure to different chemical solutions.13, 14

Functionally, it is known that newborn rats

exhibit three facial reflexes to taste. Thus, a

sweet stimulus applied on the top of the

tongue starts a reaction that may be

delightfully, because the pup licks its lips and


4

moves its tongue laterally without showing an

aversive facial expression. On the other hand,

the application of a bitter or sour stimulant

initiates a displeasure reaction with head

drawback movements, torsion of the neck and

tongue, and active mouth movements. A salty

stimulation causes a facial response

intermediate between those mentioned

above.15, 16

Investigations made by Hall and Bryan,13

using the facial reflex evoked by a taste

stimulant applied on the back of the tongue,

showed that the young clearly distinguished

between water and sucrose when they were 3

days old, although the full facial response was

not obtained until postpartum day 6. More

recently, it was observed that the oral infusion

of citric acid or quinine in 1-day-old rats

elicited an aversive or reduced response,

respectively; however, certain patterns of the

adult rat’s general stereotypical behavior (chin

scratching, mouth opening, and body

movements) did not appear in rats until 12

days after birth. These findings do not mean

that before day 12 they cannot detect the

gustatory cues of the solutions. Instead, the

fact that they do not show such an efficient

response may reflect the immaturity of the

neuronal substrates that regulate the motor

control of this aversive response.17, 18

Thus, newborn mammals can respond in a

different way to basic flavors, and this response

varies with the CNS developmental stage. For

example, the response to a salt cue has been

studied in rats from 3 to 18 days old by placing

a catheter into the oral cavity to provoke a

facial response. Rats, ranging from 6 to 18 days

of age, showed a U-shaped curve of response

with time, where the newborn has a high initial

preference for salt that gradually declines over

the following weeks, and then returns in the

adulthood. According to several authors this is

clear evidence that discrimination through the

gustatory system already occurs with a pre-

functional activity very soon after birth.11-19 To

study the sucrose-induced appetite ontogeny,

rats from 3 to 15 days old were implanted with

and stimulated via an oral cannula through

which different concentrations of sucrose and

polycose (0.03 M and 0.3 M, respectively) were

applied. After assessing the general motor

activity, it was concluded that the gustatory

discrimination between 0.3 M sucrose and

water is achieved at 6 days and between the

polycose solution and water at 9 days of age.20

Using the same experimental paradigm as for

sucrose and salt, it has been shown that taste

discrimination between water and quinine is

already present at birth, although a consistent

response is not observed until 9 days of age.

Likewise, the stereotypical reaction to quinine

seems to be shown at 12 days after birth, and

by 15 days it can be used to discriminate taste

preference or aversion in the young.15, 18

Thus, it is possible that the three motor

mechanisms for facial expression are present at

birth, although they are not yet fully developed,

this is the concept of prefunctionality

previously described to chemosensory systems

described elsewhere.21 These findings suggest

that the gustatory system is well organized and

functionally active before the structural

development of the gustatory papillae is

completed.

2. Taste bud development

The gustatory system is regulated by

specialized receptive cells that are organized in

groups of 50-100 cells, forming a spherical

structure named the taste bud (Figure 1). The

taste buds are located in three different types

of gustatory papillae on the surface of the

tongue: the circumvallate papillae located in the

medial and posterior zone of the tongue and

made up of hundreds of taste buds, the foliate

papillae located on the lateral posterior area of

the tongue where there are hundreds of buds,

and the fungiform papillae distributed over the

front two-thirds of the tongue's surface and

which usually contain one taste bud (Figure 1).

There are also taste buds on other structures

of the oral cavity, such as the soft palate and

the naso-incisor duct.22, 23

In the rat, the formation of the

circumvallate and foliate papillae is initiated

around gestational days 14 and 15, when the

epithelium covering the tongue invaginates into

the mesenchyme, and the nerves can be

observed at the center of the circumvallate

papillae on gestational day 16. On day 20 of


5

gestation the immature buds can be clearly

identified morphologically.24-27 As development

proceeds, the papilla epithelium is taking shape,

while the slot that shapes it becomes wider,

and more taste buds appear in this area. In

mammals, the morphological and functional

study of receptors located on the tongue

allows us to recognize the heterochronic

development of receptors in the oral cavity.

Figure 1. A). Taste buds consist of different types of small cells involved in a morphofunctional recycling

process. The taste receptor cells, contains microvilli that project into the taste pore and contains the sites

for sensory transduction. Basal cells derived from the surrounding epithelium that is in an initial phase of

changing to taste receptor cells. The cells are continuously in a renewing process. B). Taste buds are

contained within three major classes of papillae. Circunvallate papillae in the rat are unique structures placed

in the posterior part of the tongue that contain receptors to sour and bitter stimuli and contain

approximately 400 taste buds; foliate papillae are located at the border of the posterior tongue and are

mainly responsive to sour stimuli and contain around 100 taste buds and fungiform papillae are located on

the most anterior part of tongue and they are primarily sensitive to sweet and salt cues and contain only

one taste bud. C) Surface to the tongue rat that show the distribution of the different papillaes (Modified of

Munger, 2006).

As a general rule, a taste bud reaches full

maturation when a gustatory pore appears in

its apical part. This pore is the link between the

chemical substances contained in foods and the

internal medium. At birth, the taste buds of the

soft palate (SP) and of the fungiform papillae

(FP) are partially mature; by contrast, in buds

of the front part of the tongue, some gustatory

pores are not observed until the second

postnatal week.28 At birth the rat has

approximately 127 taste buds in the SP, but

only 53% of them have a gustatory pore. In the

case of the FP 110 buds were observed, but

only 14% of them had a gustatory pore. At the

end of the first postnatal week the number of

buds in the FP increases rapidly, and 90% of

them have pores, while 80% of the buds in the

FP have pores at this time. In the foliate (FoP)

and circumvallate (CP) papillae, some taste

buds with pores appear during the second

postnatal week when 52% of the 132 taste

buds have a pore (Figure 2). In the rat at early

postnatal stages, the fastest addition of taste

buds clearly occurs during the early postnatal

stages.29, 30, 22

The taste buds are in a continuous cycle of

regeneration due to a local phenomenon of

death and differentiation of the taste receptors

throughout the animal’s entire life.31 The role

of this cyclic death/regeneration of taste buds

and their reinnervation under normal and

pathological conditions is, at present, poorly

understood. In addition, it is still not known

how gustatory information is modulated or

how it influences brain development.


6

Nevertheless, the plastic gustatory properties

of the early brain may bypass the taste bud-

recycling process in order to maintain

homeostatic fluids and food-intake balance.

Figure 2. Number of taste buds at different ages of

rat's development. SP, soft palate; FP, fungiform

papillae; CP, circunvallate papillae, and FoP, folliated

papillae. (Modified of Harada et al., 2000).

From other studies it is known that the

maintenance and differentiation of the taste

buds in the adult rat depend on afferent

innervations and that the interaction among

specific nerves and areas of the tongue

epithelium regenerates the buds.32-35 The

gustatory nerve endings release trophic factors,

activating a program that promotes basal cell

differentiation in the epithelium of the

gustatory papilla.23

3. Development of CNS afferents and early

properties of the gustatory system

Several studies indicate that all morphogenetic

events that characterize the appearance and

maturation of the buds and their neuronal

afferent elements influence gustatory function.

Thus, at the peripheral level, changes can be

expected in the expression and regulation of

transduction mechanisms and in the

development of afferent responses.36

Previous electrophysiological studies have

analyzed the ontogeny of the electrical

gustatory responses in the afferent fibers that

convey the information from the taste buds to

the brainstem STNr. Thus, when the electrical

activity elicited by a sweet oral cue is recorded

from the tympanic chord (TC) and compared

to the magnitude of the reference response

provoked by a 10 M NH4Cl solution that does

not change with the age, the responses for

glucose and fructose do increase significantly

with age. In 14- to 20-day-old hamsters, the

responses to glucose and fructose are

significantly smaller than those in adults, and in

addition, the magnitude of the response

measured at 25 to 35 days old is intermediate

between those of newborn and adult subjects.

When compared, the response of the neuronal

system to monosaccharide and polysaccharide

showed that the sensitivity to monosaccharide

gradually increases during postnatal

development, whereas the response to

disaccharide rises more sharply at the end of

this period.37 In general, these results show

that the response properties of the TC mature

in a different way, with the response to

monosaccharide appearing earlier than the

response to more complex sugar compounds.37

On the other hand, the responsiveness of the

TC could be related to the postnatal changes in

the intracellular membrane components

involved in the transduction of the gustatory

stimulus. The age-related changes in the TC

response to NaCl and LiCl are largely

attributed to changes in the sensitivity of

individual fibers to salts. Approximately 90% of

the TC fibers in 14- to 20-day-old rats respond

to 0.10 and 0.5 M NaCl and LiCl; in addition,

the average frequency of response to NaCl or

LiCl increases about two-fold more than that

to NH4Cl. However, when the TC fibers were

classified according to the salt to which they

best respond, the number of fibers that are

sensitive to NaCl and LiCl rises sharply

between the neonatal and adult period. During

that same period, there is a reduction in the

number of fibers with a preferential response

to NH4Cl. Therefore, the increase in TC

sensitivity to NaCl and LiCl during

development may be due to an increase in the

ratio of fibers that respond more strongly to

NaCl and LiCl, as well as to the increased

sensitivity of individual fibers to these

stimulants.38

Specifically, the TC electrical responses to

NaCl and LiCl in 13- and 23-day-old rats is not

significantly affected by lingual pre-treatment


7

with 100 M amyloride (a substance that

promotes membrane input resistance). By

contrast, in rats that are 29-31 or 90-100 days

old, amyloride suppresses responses to NaCl

and LiCl. From this study Hill and Bour39

concluded that the increased sensitivity to Na+

and Li+ and to amyloride, are due to the

gradual increase in the functional expression of

amyloride-sensitive Na channels in the apical

membranes of the gustatory cells.

The changes associated with the role of

Na+ during the development of taste have also

been documented in mice, rat, hamster, and

sheep. Bradley and Mistretta25 showed in

pregnant ewes that the TC responds to a

variety of gustatory stimuli. Later, it was also

shown that TC sensitivity to LiCl and NaCl in

sheep increases progressively during pre- and

postnatal development.40 The increased

gustatory sensitivity to Na and Li+ was

attributed by these authors to changes that

depend on the age of the gustatory cells with

apical membranes that contain functional

transduction systems, which were recognized

later as amyloride-sensitive Na+ channels.41

However, it is still unclear what modulates the

gustatory response to these salts. The work of

Hill and Bour39 showed that the increased

sensitivity to NaCl and LiCl occurs in parallel

with an increased sensitivity to the inhibitory

effects of the amyloride.

Immunohistochemical techniques were used

to demonstrate that amyloride-sensitive Na+

channels are present in 2-day-old rats and that

they are located in the apical membrane of the

gustatory cells; the next question was: “Are

they functional?” Mc Pheeters et al.,42 reported

that Na+ currents sensitive to amyloride are

present in approximately 40% of gustatory cells

isolated from the FP of 2-day-old rats, and that,

following a dose of 30 M amyloride, the input

resistance of the membrane significantly

increases. However, apical sensitivity to

amyloride is not apparent until later in

development, and amyloride-sensitive Na+

channels may be present in the basolateral

membrane of neonatal gustatory cells. This

distribution is consistent with the

immunoreactivity for Na+ channels in the

basolateral membrane that is observed in the

FP gustatory cell membranes.43

The temporary discrepancy between the

appearance and function of the buds and the

expression of their sensitivity to amyloride

suggests that, after birth, endocrine and

exocrine events may activate the development

of previously quiescent Na+ transport or

transduction of Na+ signals in the rat’s

gustatory system.43 Therefore, the morphology

of taste buds constitutes the basis for

understanding the changes in the response

properties of the gustatory peripheral system.

Recent studies attempt to identify the

mechanisms that regulate these changes, and

they focus on specific, G-protein-coupled

membrane receptors related to sweet and

bitter solutions that transduce the gustatory

stimulation into mechanisms or signals that

regulate the development of the peripheral

gustatory system. Likewise, these studies also

aim to identify specific membrane receptors

coupled to G-proteins that transduce signals to

the second neuronal relays. The resulting

electrophysiological changes may reflect

alterations of the affinity or density of the

neuronal gustatory system receptors or

changes of the second messenger.44

Information regarding electrophysiological

development of glossopharyngeal and vagus

nerves is still scarce, because the stimulation

that generates the response of these nerves is

more complex. Another reason is the technical

difficulty of performing the same timeframe

study that has been made on the TC.

However, authors such as Hill37 mention that

each cranial nerve may have a fundamental

influence that can modulate the gustatory

function.

4. The first relay of the gustatory system in

the CNS

The STN is the first relay of the gustatory

system and it conveys information from

afferent axons that innervate the taste buds in

the tongue. The STN is a very complex nucleus

because at this level information is combined

related to basic respiratory, gastro intestinal

and gustatory systems necessary for newborn


8

survival in the nest. This nucleus is generated

during gestation, and at birth it exhibits rapid

neuronal growth, making it a good model for

studying the development of the plastic

properties and their age-related changes.

Figure 3. A). Schematic drawing of the central taste pathways in the rat. VII, facial nerve, IX,

glossopharyngeal nerve, X, vagus nerve; STN, solitary tract nucleus; RF, reticular formation; PBN,

parabrachial nucleus; UZ, uncertain zone. LH, lateral hypothalamus; Am, amygdala; PMV posteromedial

ventral thalamic nucleus, GC, gustatory insular cortex (Modified of Yamamoto et al. 1998). B) Schematic

representation of the dorsal surface of the medulla indicating the different areas of the nucleus of solitary

tract. STNr, i and c; solitary tract nucleus rostral, intermediate and caudal. C). Horizontal subdivision of the

STNr showing the subnuclear organization, t, solitary tract; M, medial subnuclei; CR, rostral central

subnuclei, RL, rostral lateral subnuclei and V, ventral subnuclei.

4.1 Anatomical characteristics of the STN

The STN is located in the bulbar area of the

brainstem (Figure 3). Taking the vertex of the

head as reference it lies in the rostral portion

and at coordinates –10.52 to 13.24.45 The

somatic sensitive column of trigeminal and

glossopharyngeal nerves is adjacent to the

STN; the vestibular lateral nerve as well as the

vestibular medial nerve are dorsal, and the

reticular parvocellular nucleus is ventral to the

STN.

In the adult rat, the STN is considered an

integrative station of very complex

information, and for research purposes it has

been divided into three main areas: the most

rostral part, denominated the STNr, receives

special visceral afferent information from the

gustatory receptors in the tongue and

epiglottis and conveys it to the facial,

glossopharyngeal, and vagus nerves. The

intermediate (STNi) and caudal (STNc) areas

receive information from the cranial

glossopharyngeal (IX), vagus (X), and trigeminal

(V) nerves, which are responsible for the

general visceral afferents, including information

from chemoreceptors (IX), baroreceptors (X),

pulmonary distension receptors (X), intestinal

receptors (X), and mechanoreceptors (V).46-50

The gustatory portion of STNr can be defined

electrophysiologically by the location of

neurons that respond electrically to taste

stimulation or anatomically by distributing

neuronal axonal branches that convey

gustatory information. The gustative area is

expanded from the rostral end of the STNr to

the medial edge of the nucleus as far as the

fourth ventricle (lateral 2.72 mm).45

Recent studies have shown that the STNr is

made up of 4 sub-nuclei that are named with

reference to the solitary tract (ST), which

crosses exactly over the center of the nucleus

from the caudal to the rostral portions. The

sub-nuclei that constitute this structure are:

the central rostral (CR), the lateral rostral

(LR), the ventral (V), and the medial (M)

(Figure 3).50

The CR sub-nucleus contains neurons that

receive information from taste buds whose


9

peripheral afferent fibers convey information

via the glossopharyngeal, facial, and major

superficial petrosal nerves. Studies using

neuronal stains have shown that neurons of

this area relay gustatory information, since they

send axonal ascending fibers and information to

the next neuronal relay, the parabrachial

nucleus (PBN). The LR subnucleus is the main

site of tactile input, which is carried by the

trigeminal nerve that innervates the taste buds

of the tongue, bringing somatosensorial

information to this portion of the nucleus. Sub-

nucleus V is the main origin for STNr

projections to motor centers in the brainstem

such as the facial motor nucleus, the

glossopharyngeal, and the vagus dorsal motor

nucleus. The M sub-nucleus plays an important

role in intra-nuclear communication between

the caudal and gustatory areas, suggesting that

the information coming from the external

medium into the gustatory area interacts with

that generated in the internal medium (Figure

3).50

4.2 STN afferents and projections

Gustatory receptor activity is transmitted into

the brainstem along three cranial nerves. One

is the tympanic chord, which is an anastomotic

union between cranial nerves VII and VIII that

gather the information from the front two-

thirds of the tongue, and specifically, from the

fungiform gustatory papillae, from a population

of small buds in the buccal wall of the

sublingual organ, and from some of the foliated

papillae. The second is the glossopharyngeal

nerve, which transmits the information of the

back third of the tongue, coming from the

circumvallate gustatory papillae and the rest of

the foliated papillae. The third is the vagus

nerve, which gathers the information from the

epiglottis, part of the palate, and the upper

portion of the esophagus.51, 52

The first order neurons gathering the

different gustatory modalities originate in the

papillae and have their cell bodies in the

peripheral ganglia: the geniculated, petrosal,

and nodose ganglia located at the cranial cavity

entrance. The central branches of these

ganglionic neurons penetrate the brainstem at

the bulb level where they make the first

synaptic contact with the STNr neurons.53

The STNr efferent neurons in rodents

project ipsilaterally to the PBN dorsal middle

area at the pontine level, where a topographic

layout in this nucleus has also been described53

The PBN efferents project ventrolaterally to

the uncertain area over the internal capsule to

connect with the ventral portion of the

forebrain, to the central nucleus of the

amygdala, to the red nucleus, and to the

terminal groove. Other neurons project

ipsilaterally to the thalamus as far as the ventral

posteromedial nucleus, and the thalamic

neurons project to the agranulocytic part of

the insular cortex near the zone of the tongue

(Figure 3).

Some of the STNr neuronal axons cross to

the opposite side near the thalamus and the

pontine area, giving a counterlateral character

to the gustatory tract. Like other nuclei

involved in the gustatory tract, ascendant

neuronal relays also show a “taste-topic”

distribution of the gustatory information.54

4.3 Types of neurons in the STNr

The STN is a reticular-shaped structure

formed by different types of neurons. Knowing

the cell types of any structure helps to

understand the relationship between the

morphology and the function of the cells of

neuronal circuits. Diverse methodological

strategies have been used to classify the STNr

cells. However, the staining strategies that have

been used (Nissl, biotin, Golgi-Cox, and rapid-

Golgi procedures) to visualize the shape, size,

orientation, dendritic distribution, projection

site, etc, are still controversial. These stains

can determine the morphology of cells in the

STN but cannot show whether these cells

respond to gustatory stimulation or how

differences between cells may affect the

functional properties of the gustatory

response. In order to solve this problem,

electrophysiological studies were made to

identify which neurons respond to chemical

stimulants placed on the back of the tongue;

responsive neurons can also be marked by


10

using neurobiotin, which allows visual

recognition of the cell morphology.55

The cell types which have been described

by these techniques are: multipolar neurons,

which are triangular or polygonal in shape with

3 to 5 primary dendrites; fusiform neurons,

characterized by an elongated soma and two

main primary dendrites arising at opposite

poles; and small ovoid neurons having 2-4 thin

primary dendrites (Figure 4). Using the Nissl or

Golgi techniques, multipolar neuronal

subgroups can be identified, and these are

subdivided into large, small, and ovoid shapes

which have a similar subdivision.54, 56-61

Figure 4. Photomicrography of multipolar (M);

ovoid (O) and, fusiform (F) neurons of the STNr,

stained with the Golgi-Cox technique. Calibration,

50 m.

The most common neurons in the STNr are

ovoid (63%) and fusiform (19%), and the

remaining 18% are multipolar.58 Based on

techniques with neuronal tracers, multipolar

and fusiform neurons have been observed

projecting to the PBN.54, 57 Likewise, there are

multipolar neurons in the ventral part of the

STN that send information to the reticular

formation and to the motor nuclei of the

cranial nerves (V, VII, IX, X, and XII).62-64 It has

been suggested that the neurons projecting

rostrally are involved in the processing and

relaying of gustatory information. Meanwhile,

the caudal projections are thought to be

involved in the reflex control of saliva

secretion and food ingestion.64 Ovoid neurons

are believed to be local, interconnecting

interneurons that modulate the nucleus

output.57

4.4 Neurogenesis and neurochemical

development of the STN

The STN layout in adult mammals has been

widely studied,65-69 but to our knowledge there

is little information about the development of

the morphofunctional properties of this

structure. Studies of STN development are

important, because the neuronal information

comes from critical areas that are

physiologically necessary for newborn survival,

such as the respiratory, cardiovascular, and

digestive systems.70

The first ontogenetic studies were made by

Altman and Bayer71 using the 3H-thymidine-

radiographic technique. They reported that

neurons reaching the STN are generated

between gestational day 11 (E11) and E14, with

a peak of neurogenesis on E12. From recent

studies it is known that at birth, the primary

afferents to the STNr are organized in a

viscerotopic pattern equivalent to the adult

stage. The afferents of the facial and vagus

nerves reach the STNr by embryonic day 17

(E17), and on E19 they show a mature,

organized pattern.70 In the case of the

glossopharyngeal nerve there is a controversy,

since Lasiter72 mentioned that in the rat the

glossopharyngeal does not reach the STNr

until 9 or 10 days after birth. Recently, the

Zhang’s group was unable to mark the

glossopharyngeal nerve during the embryonic

period because of its proximity to the vagus

nerve; they suggest that it may follow a

developmental pattern similar to that of the

facial and vagus nerves, but this has not yet

been demonstrated. These differences may be

due to the anatomical techniques used by the

two groups. The afferents of most of the

information sources to the STN are well

represented before the last differentiation

period (E17 to E19), when the chemical

properties are established.70 It is possible that

the input from these afferents may be

responsible for triggering the rapid STN

differentiation.


11

Immunohistochemistry and histochemical

studies of neurochemical development have

demonstrated the presence of

acetylcholinesterase in the STN between E15

and E17. Immunoreactivity for calbindin and

calretinin appears in later stages of gestation

with a peak at postnatal day P10. Neuron

immunoreactivity for tyrosine hydroxylase was

recognized on E15, showed rapid

differentiation on E17, and reached the adult

pattern on day E19. Immunostaining for

substance P showed an adult distribution

pattern on E19.70

These results indicate that the patterns of

immunohistochemical development

differentiate rapidly between E15 and E17,

remaining more stable by E19. This suggests

that the morphologic and chemical features of

the STN are present even before birth; thus,

the nucleus is prepared to be involved in its

vital functions at the time of birth.5, 6, 70, 73-75

The peaks that are observed for each

marker may represent an accelerated

development of the connections and essential

circuits in the STN associated with the primary

necessities for postnatal survival (for instance,

respiratory, cardiovascular, and digestive tract

functions). Furthermore, the postnatal peaks

may suggest additional elements that are

required to establish paths with essential

connections, or formation of routes related to

non-essential behaviors during postnatal life,

for example in the gustatory system, the plastic

changes underlying the switching of pups from

liquid to solid food intake.

4.5 Synaptogenesis in the STNr

Although some synaptic buttons have been

observed on E17, these structures in the

afferents to the STN develop mainly on E19,

followed by the beginning of chemical

differentiation as described by Zhang and

Ashwell.69 This developmental period

corresponds to the initial architectonic

differentiation of the STN. It is possible that

the maturation of the synaptic terminals may

be related to the neurophyllum organization.

The gustatory glomeruli are highly

preserved units constituted by the afferents

that convey information into the STN neurons

from different brain sources, including the

pulmonary, laryngeal, and taste afferent

fibers.48, 70, 76, 77 Zhang and Ashwell69 did not

observe any taste glomeruli during the

embryonic period or the first weeks of life in

the rat. Therefore, they speculate that at birth,

most STN primary functions, including

cardiovascular control, may be modulated by

simple circuits that have not matured to form

synaptic glomeruli. The postnatal development

of the synaptic glomeruli may lead to numerous

changes in the layout of STN connections,

which may be modified in accordance with

postnatal needs and the plasticity of the

organism.69 The glomeruli units, whose

formation and function begin prenatally and

whose maturation is complete after the first

postnatal weeks, are a feature of the adult

stage and are used for chemosensory functions.

In this regard, the pre- and neonatal STN

functions may be regulated by means of

axodendritic afferent signals from the gustatory

receptors to the STN neurons.

4.6 Neurotransmitters involved in STN

function

Neurotransmitters and their precursors in the

STN have been identified by immunostaining

techniques, revealing that both neurons of the

peripheral ganglia where the cellular bodies are

located and the neurons carrying information

into the STNr contain substance P, tyrosine

hydroxylase, vasoactive intestinal polypeptide,

calcitonin gene-related peptide, galanin,

glutamate, and aspartate.78-82 It is not

surprising that glutamate and GABA are found

as neurotransmitters and neuromodulators in

the STNr.83 Glutamate is released from

gustatory afferents,84 and it is also contained in

STNr neuronal bodies and in some of the

neuronal projections to the PBN.85

Immunostaining for GABA can also be

detected in the STNr, mainly in the small ovoid

neurons, which are thought to be inhibitory

interneurons.56, 86, 87

Through retrograde labeling techniques, it

has been shown that dextran injection into the

central nucleus of the amygdala (CeA) labeled


12

fibers ending at different locations in the

STFNr,88 in the medial, central, and ventral part

of this structure.89 It also was found that after

the injection of cholera B toxin into the STN,

many cells in the central amygdala are labeled.

Generally, the influences of the descending

fibers to the fore brain are excitatory.

However, it has been shown that these

amygdala fibers have an inhibitory effect in the

rat.90-92 In the rat there is evidence that this

projection is GABAergic, suggesting that in

some way it may be modulating primarily local

connections, with less effect in the tract that

carries the gustatory information to upper

neuronal relays.93

The gustatory process may be modulated by

descending information from different nuclei of

the fore brain as a result of sensorial

experience. Opioid receptors, which receive

information from central amygdala neurons,

are also expressed in the STNr, suggesting

another possible modulation of the gustatory

information in the STN. Also found in the STN

are receptors for oxytocin and the

catecholamines that come from the

hypothalamic structures and possibly modulate

the hedonic aspects elicited by taste cues.92, 94-

96

4.7 Gustotopic organization in the STN

In mammals, most parts of the CNS are

constituted by maps that represent the

receptor layout. These maps in the cortex, as

in other parts of the brain, arise during

ontogeny as a result of interactions between

numerous factors. Several behavioral

investigations indicate that altricial mammals

have a functional gustatory system at the time

of birth, before the neuronal substrates attain

full anatomical maturation. The gustatory

system develops during gestation and acquires

a well-advanced organization in the days just

prior to birth, then passes through a neonatal

period of rapid development. The most evident

feature of this system is the receptor layout in

different parts of the tongue.

In mammals the somatosensory cortex is

organized according to the location of the

receptors over the body surface; this

representation seems to arise during

development as a result of experience or local

factors that participate to different extents. It is

also known that the cortical representation is

retained in other subcortical relays. This

conclusion comes mainly from studies of

electrical stimulation of the somatosensory and

motor cortex and from cortical lesions.97, 98

More recently, such investigations have been

extended to the auditory and visual pathways,

and the results are very similar to those in the

somatosensory and motor cortical

representations.99-100 Currently, the gustatory

pathway is considered an important model to

study the anatomical and functional

organization of the chemosensory systems.

Electrophysiological studies show that the

nuclei involved along the gustatory tract

maintain an organization in response to taste

stimulation.50

Immunohistochemistry techniques that

detect expression of early genes such as c-Fos

in response to specific stimuli have been used

to show the topographic organization in the

olfactory, somatosensory, and visual system

areas. The first studies using these techniques

in the gustatory system showed that sucrose

and quinine induced c-Fos expression in the

STNr, with expression greater in the medial

part of the nucleus in response to quinine, and

greater in the lateral part in response to

sucrose.101-103 Recently, a group of investigators

sought to identify the specific area that induces

c-Fos in response to quinine and to determine

the extent to which its expression is modified

by the intensity of the gustatory cue. After

application of quinine at three concentrations,

immunostain was again observed in the STNr

medial area, and it was similar at all three

concentrations.104

In similar experiments, it was found that the

STNr lateral area responds to 0.1 M citric acid,

and by means of a correlation analysis, it was

determined that c-Fos is expressed in

completely different areas for quinine and citric

acid. The data showed that the correlation of

c-Fos expression with location at the different

quinine concentrations is very high (between

0.95 and 0.99), whereas between quinine and

citric acid it is much lower (0.29), suggesting


13

that the expression areas for these stimulants

are different. On the other hand, when

applying 0.3 M NaCl and counting the

distribution of c-Fos immunoreactive neurons

in the STNr, the number of stained neurons is

quite similar to or lower than number labeled

when water was applied. Areas labeled in

response to NaCl and citric acid showed a

higher correlation (0.84) (Figure 5).104

Figure 5. Distribution of immunoreactivity for c-Fos in the STNr after a period of 45 min. of different

gustatory exposures. Calibration, 200 m. Extracted from (Travers et al., 2002).

These findings indicate that water may serve

as a control stimulant, since it is an insipid

stimulation that maintains the fluidity

characteristics of other cues. They also show

that cells marked in the STNr after stimulation

with water are cells that respond to mechanical

stimulation applied to the back of the tongue. It

is noteworthy that NaCl does produce lower

c-Fos expression, which suggests that not all

cells of the STNr respond to a specific

stimulation; this has also been shown in the

somatosensorial system, since it has been

observed that signals that travel along small-

diameter adherent fibers induce more Fos


14

expression than signals following a more

complex axonal system. This suggests that Fos

expression varies with the complexity of the

neuronal connections.105

5 Early environmental influences on the

gustatory system

In many sensorial systems, the normal function

and morphologic maturation along the

ascending neuronal relays depend on the

appropriate type of stimulation and the

selective experience obtained during well-

defined developmental periods.30, 106 It is also

important to highlight the information obtained

about the underlying processes that are

necessary for normal development.

In the literature there is abundant

information on the somatosensory, auditory,

and visual somatotopic cortical organization

obtained mainly from experiments of local

electrical stimulation of peripheral

receptors.107, 108 Unfortunately, little is known

concerning the effects of perinatal sensory

stimulation and specifically in the gustatory

system. The normal ascending patterns of

sensory information are crucial for the

establishment and maintenance of adequate

connectivity patterns and for the integrative

processes taking place at the neuronal

levels.108-109

5.1 Alterations in the STNr by sodium

restriction during gestation in the rat

In relation to the effect of sensorial stimulation

on the gustatory pathway, it is known that Na+

restriction (0.03% NaCl in the diet), starting on

day 8 after conception and continuing

throughout development, noticiably reduces

the neurophysiological response to NaCl in the

tympanic cord. This response is reduced in

more than 60% in the restricted animals

compared to controls whose diet was normal

(approximately 1.0% NaCl). For comparison,

the TC responses to NH4Cl and other

stimulants were not affected by Na+

restriction in the maternal diet.110 The same

authors reported in 1991 that NaCl

deprivation influences TC terminal fields and

the STNr. Thus, the groups that were under

sodium restriction showed irregular and larger

shapes in TC terminal fields than the controls,

and even after restrictions longer than 60 days,

restoring NaCl to the diet can reverse the

damage at the TC level. However, other

studies indicate that the functional nerve

recovery is not sufficient to promote

anatomical restoration.111, 112

The lack of neuronal information reaching

the TC during development may contribute to

the neurophysiological changes observed in this

structure, since the activity produced by

sodium administration is essential to form an

adequate terminal field.59, 112

5.2 Alterations produced in the STNr by

perinatal undernutrition

Regarding the effects of sensory and food

intake restriction on the gustatory sensorial

channel development, the available information

is scarce. In particular, the question, to what

extent the effects of malnutrition may influence

the anatomic and functional organization of the

STN, has been ignored, in spite of the fact that

it is one of the most significant relay areas of

the brainstem on the route of neural impulses

to the cerebral cortex.57, 110

When neonatal food is restricted during

periods of rapid brain development, the

presence of taste substances in the mouth is

significantly reduced, causing (not only the lack

of food, but also) a decrease of gustatory

stimulation. Similar conditions of reduced

content will prevail in the rest of the digestive

tract, with possible consequences of reduced

afferent information reaching the caudal and

intermediate STN regions.

Using the model of perinatal food

restriction in rats at different gestational ages

by reducing the food intake of pregnant

females, and neonatally by placing pups for 12 h

of each day with a nipple-ligated mother and 12

h with a normally lactating mother, we found

that in the malnourished group, the STNr

neurons become hypotrophic compared to the

controls. Furthermore, interneurons showed

fewer and shorter dendritic prolongations. In a

rehabilitated group with restricted food before

birth but normal food intake during the


15

lactating period, the neuronal morphology was

similar to that of controls.113

Prenatally malnourished subjects, fed and

cared for by a pair of normal “wet nurse”

mothers (rehabilitation), revealed interesting

aspects of STNr neuronal plasticity. Thus, the

finding of a larger number of branches in the

distal parts of the dendritic trees of STNr

neurons contrasts with the result obtained in

malnourished animals with no postnatal

rehabilitation. On the other hand, in prenatally

malnourished groups either with or without

rehabilitation, the dendritic extensions are

larger in the distal portion of the dendritic tree

than in controls, suggesting a possible

compensatory mechanism of a plastic nature.

This interpretation is supported by the

“covering” and “tiling” phenomena by which a

neuron’s dendrites of the same functional

group extend to cover nearby zones where

neuronal death or damage occurred in an

adjacent dendritic tree.114 Taken together, this information shows that

gustatory stimulation in early stages of life is

necessary to induce normal neuronal

development of the STNr.71-113 Later, during

the lactating period it may accelerate taste bud

development and promote neuronal

maturation.115

II. Conclusions

The experimental findings included in this

review allow us to appreciate the vast scope of

the field of gustatory physiology; technological

progress has generated original and novel

information that has been used to identify new

basic neuronal mechanisms of chemoreception.

For instance, now it is undeniable that the

uterine environment is an important source of

sensorial experience for the fetus and that the

gustatory and olfactory signals from the

amniotic fluid contribute to prenatal brain

development. It is also evident that in altricial

species, neuronal substrates are already

precociously developed at birth in order to

satisfy the basic needs and survival of the

newborn. The time of birth is the critical stage

for obtaining early experience and plastic

capabilities of brain tissue to be used later in

life.

Another important contribution to the

knowledge in this field is the developmental

characterization of gustatory afferents, since

this allows appropriate timeframes to be

selected for a specific study of the structures

involved in gustatory signal transduction, such

as ion channels and receptors. This

characterization also determines the

correlation between the afferent connectivity

and the specific neuronal activation by different

chemical compounds at critical ontogenetic

stages of the gustatory pathway.

The gustotopic organization is another

important line of research that has been

studied recently in order to determine

whether the neuronal relays are anatomically

and/or functionally organized for the different

basic flavors (sweet, salty, sour, and bitter). As

a result of these investigations, it has been

suggested that in the STN, the cell layout that

responds to basic flavor is segregated, and it

may be related to the hedonic and behavioral

characteristics resulting from stimulation by

each flavor. On the other hand, it has been

shown that not only the information from the

stimulation of the oral cavity receptors by

chemical stimulants, but also information

coming from other sources (somatosensorial

and visceral) has a complex influence upon the

STNr neuronal substrate. These findings

indicate that the basic mechanisms underlying

the taste sensitivity for food intake are also

operating during harmful or aversive food

rejection as a part of the early gustatory

experience.

Current studies seek to define the early

stages when the STN gustatory layout is

established and to determine if they can be

altered by exposure to different epigenetic

factors. It will also be important to discover

how the dietary change from breast milk to

solid food causes anatomical and functional

changes of the plastic brainstem taste

organization. This will help to establish the

activation time of STNr neuronal sensitivity to

basic flavors and critical ages for neuronal and

anatomical organization, and to study the

plastic neuronal properties associated with

chemoreception in both normal and altered

perinatal conditions.


16

III. Acknowledgements

The preparation of this manuscript was partly

supported by a grant from DGAPA/UNAM,

IN207310-21. We thank Dr. D. Pless for

editorial assistance, and N. Hernandez for

image analysis.

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Recibido: 8 de marzo de 2011

Aceptado: 9 de mayo de 2011













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